Cardiovascular diseases are responsible for a preponderance of health problems in the majority of the developed countries as well as in many developing countries. Heart disease and stroke, the principal components of cardiovascular disease, are the first and third leading cause of mortality in the U.S., accounting for nearly 40% of all deaths (Heart and Stroke Statistical Update, American Heart Association 2002). Cardiovascular diseases also include congenital heart defects, which occur in about 1% of live births (R. F. Gillum, Am. Heart J. 1994, 127: 919-927) and are the main cause of mortality in the first year of life (J. L. Hoffman, Pediatr. Cardiol. 1995, 16: 103-113 and 115-165). When they do not lead to death, cardiovascular diseases may alternatively result in substantial disability and loss of productivity. About 61 million Americans (almost one-fourth of the population) live with cardiovascular disorders, such as coronary heart disease, congenital cardiovascular defects, and congestive heart failure. In 2001, 298.2 billion dollars were spent in the treatment of these clinical conditions, and the economic impact of cardiovascular disease on the U.S. health care system is expected to grow as the population ages.
Over the past 30 years, advances in the treatment and prevention of cardiac diseases have led to constantly declining morbidity and mortality rates. Treatments for both congenital heart defects and cardiomyopathies have become more sophisticated. However, when these treatments fail, organ or tissue replacement remains the only other possible option. Different surgical procedures may be performed to treat heart failure and cardiac deficiency. These procedures include transplantation of organs from one individual to another, reconstructive surgery, and implantation of mechanical devices such as mechanical heart valves.
Cardiac transplantation is so common that the primary limitation on patient outcome is not the surgical technique, but the declining availability of donor organs. In 2000, 2,500 heart transplants were performed in the U.S. while it was estimated that between 20,000 and 40,000 patients could have benefited from such a medical operation. To circumvent the problem of donor organ scarcity, one can resort to surgical reconstruction, whereby damaged or defective tissue at one site of the patient is replaced by healthy tissue from another part of the patient's body. These autografts include blood vessel grafts for heart bypass surgeries. The disadvantages of using autografts are their limited durability (E. Braunwald, Heart Disease 4th Edition, E. Braunwald (Ed.), W. B. Saunders: Philadelphia, Pa., 1992, pp. 1007-1077) and a loss of function at the donor site. In addition, reconstructive surgery often involves using the body's tissues for purposes not originally intended, which can result in long-term complications. Mechanical heart valve prostheses have proved to effectively improve patient's quality of life. However, they are also subject to mechanical failure and rejection, can induce inflammation and/or infection, and require long-term drug intervention to prevent blood-clotting. Furthermore, since these mechanical valve substitutes are nonviable, they have no potential to grow, to repair or to remodel; therefore their durability is limited, especially in growing children (J. E. Mayer Jr., Semin. Thorac. Cardiovasc. Surg. 1995, 7: 130-132). Since currently available treatments (with the exception of cardiac transplantation) are only palliative, new drugs and procedures for treating cardiovascular diseases, especially approaches allowing the recovery of diminished cardiac function, are highly desirable.
Tissue engineering is emerging as a significant potential alternative or complementary solution. In tissue engineering, tissue or organ failure is addressed by implanting natural, synthetic, or semi-synthetic tissue and organ mimics that are fully functional from the start or that grow into the required functionality to replace, repair, maintain and/or enhance organ/tissue function. Although efforts to generate bioartificial tissues and organs for human therapies go back at least thirty years, such efforts have come closer to clinical success only in the last ten years. In addition to developing improved bioartificial tissue equivalents for therapeutic purposes as well as for in vitro research and drug development, tissue engineering also aims at providing measures to enhance survival and integration of engineered grafts following implantation in vivo.
One of the major strategies adopted for the creation of engineered tissues is the in vitro growth of isolated cells on three-dimensional templates or scaffolds under conditions that coax the cells to develop into a functional tissue. The scaffolds, which can be fashioned from synthetic polymers or from natural materials such as collagen, temporarily provide the biomechanical support needed by the cells. As the cells grow and differentiate on the scaffold, they produce their own extracellular matrix. When implanted, the bioartificial tissue should become structurally and functionally integrated into the body.
The feasibility of engineered functional cardiac muscle has been demonstrated (T. Eschenhagen et al., FASEB J. 1997, 11: 683-694; L. E. Freed and G. Vunjak-Novakovic, In Vitro Cell Dev. Biol. 1997, 33: 381-385; R. Akins et al., Tissue Eng. 1999, 5: 103-118; N. Bursac et al., Am. J. Physiol. Heart Circ. Physiol. 1999, 277: H433-H444; R. L. Carrier et al., Biotechnol. Bioeng. 1999, 64: 580-589). Eschenhagen and co-workers (T. Eschenhagen et al., FASEB J. 1997, 11: 683-694) showed that embryonic chick cardiac myocytes cultured in collagen gels displayed characteristic physiological responses to physical and pharmacological stimuli; and Akins et al. (R. Akins et al., Tissue Eng. 1999, 5: 103-118) demonstrated that rat ventricular cardiomyocytes cultured on polystyrene microcarrier beads in bioreactors formed three-dimensional spontaneously contractile aggregates. Cultivation of neonatal rat cardiac myocytes on polyglycolic scaffolds in bioreactors has been shown to result in contractile three-dimensional tissues (L. E. Freed and G. Vunjak-Novakovic, In Vitro Cell Dev. Biol. 1997, 33: 381-385) with ultrastructural features of cardiac muscle (N. Bursac et al., Am. J. Physiol. Heart Circ. Physiol. 1999, 277: H433-H444). These studies also provided evidence that variations in initial cell density and cultivation conditions affect the structure of the engineered cardiac tissue produced (R. L. Carrier et al., Biotechnol. Bioeng. 1999, 64: 580-589; M. Papadaki et al., Am. J. Physiol. Heart Circ. Physiol. 2001, 280: H168-H178).
One of the major difficulties in engineering a functional cardiac tissue starting from isolated cells is the heart's complex structure and function at different spatial scales. The complex structure of the heart stems from the elongation and spatial alignment of cardiac myocytes, from the distribution of intercellular connections, and from the formation of cardiac muscle fibers and bundles that rotate transmurally inside the heart wall. This unique architecture of cardiac tissue enables an orderly sequence of electrical and mechanical activity and efficient pumping of blood from the heart. Unsurprisingly, the intricate arrangement and geometrical order of different cell types in living cardiac muscle tissue is difficult to reproduce in vitro; and standard tissue engineering culture of cardiac muscle cells seldom yields tissue of such complex structure.
Indeed, most tissue engineering techniques used so far have led to cardiac muscle constructs with a number of shortcomings that limit their usefulness for both in vitro and in vivo applications. Most often, unlike native cardiac muscle that consists of fibers with a defined orientation, the cells in engineered constructs exhibit random orientation and a poor degree of differentiation. Furthermore, the constructs often present a non-uniform spatial cell distribution with, for example, a good tissue formation at the periphery and a loose network of disoriented cells at the center of the construct. Since only a minor fraction of the three-dimensional structure consists of cardiac tissue, its usefulness as a medical implant for replacement therapy is limited. Furthermore, it is generally recognized that structural and functional integration of engineered grafts remains unsolved. Even if high-fidelity engineered tissue equivalents become available, the process of integration will need to be enhanced for the tissue graft to survive implantation and remain functional.
Therefore, a need continues to exist for new strategies that offer novel and satisfactory platforms for in vitro research and the eventual development of compact, thick and functional transplantable heart muscle. In particular, methods for the production of three-dimensional bioartificial constructs with properties resembling those of native cardiac tissue are highly desirable, as are methods allowing their enhanced integration and functionality in vivo.